Summary Alphaviruses represent a continuingly emerging challenge to global public health. This is exemplified by the spread of chikungunya virus from Africa through southern Asia and into the Americas. Additionally, this past summer has seen an increase in the number of eastern equine encephalitis virus infections and deaths in the United States. A deeper understanding of the determinants of efficient transmission and replication is needed to identify targets for transmission interventions. In recent years it has become apparent that modification of viral RNA impacts it?s function, and the outcome of infection. The proposed research extends on published and preliminary observations that implicate a particular epitranscriptomic modification of the viral genomic RNA in the determination of transmission efficiency. This proposal investigates the novel hypothesis that host species specific modification of the packaged viral genomic RNA impacts the ability of the particle to establish infection in the next host cell, a prospect of significant consequence for alphaviruses that obligatorily cycle between mosquito and vertebrate hosts. To examine this the proposed research will: (i) employ multiple sequencing technologies to determine the methylation state of cytosine residues in the genome of particles produced in vertebrate and mosquito cells; (ii) examine the biological consequence of disrupting the methylation of the viral genome through altering the methyltransferase and/or mutating residues found to be frequently modified. The results of this work will elucidate the pro- and anti-viral effects of cytosine methylation of viral RNA, and lay the ground work for further investigation of host-specific epitranscriptomic modifications and their role in virus replication and transmission.